The Proteomics Resource at the Institute for Systems Biology provides strategic consulting and technologies in proteomics analysis for the ISB community and collaborating researchers worldwide. The ISB Proteomics Resource has cutting-edge mass spectrometry as its technology platforms including 2D linear and 3D and ion-traps; high resolution, high mass accuracy Orbitrap equipment, MALDI-TOF-TOF and hybrid quadrupole-TOF mass spectrometers; triple-quadrupole mass spectrometer; as well as nano-capillary HPLC separation systems for automated high-throughput sample introduction. As part of the Proteomics effort at ISB, we develop open-source cross-disciplinary software programs to advance bioinformatics tools to condense, analyze, and present data for large quantitative biological analyses.

Services offered:

• Experimental design consultation.
• Sample processing at bench, e.g. in-gel digests; isotopic labeling, e.g. Isotope- Coded Affinity Tags (ICAT™), and Amine-Modifying Labeling Reagents for Relative and Absolute Quantitation (iTRAQ™).
• Sample processing off-line, e.g. cation exchange High-Performance Liquid Chromatography (HPLC).
• Sample processing in-line, e.g. liquid chromatography/tandem mass spectrometry (LC-MS/MS)
• Data analysis through ISB proteomics data analysis pipeline

The ISB Proteomics Resource operates on a cost-recovery basis for its collaborations with both internal ISB and external users

Equipment and procedures:

Autosamplers:

• Dionex FAMOS micro-autosamplers
  Agilent 1100 micro-well plate autosamplers

• Agilent binary microflow HPLC pumps
  Agilent nanoflow HPLC pump

• Thermo LCQ-DECA 3D ion-trap MS
• Thermo LTQ linear ion-trap MS
• Thermo LTQ-Orbitrap MS
• Waters/Micromass Q-Tof Premier API MS
  • Sample preparation and fractionation for LC-MS/MS including:
    1. sample (protein) precipitation, resolubilization, and digestion;
    2. sample cleanup (e.g. by off-line reverse-phase method);
    3. isotopic labeling by ICAT™ or iTRAQ™ for relative quantitation of proteins in complex samples;
    4. off-line strong cation exchange fractionation.
  • High-throughput LC-MS/MS
  • Data analysis via ISB proteomics data analysis pipeline: Sequest > Peptide Prophet > Protein Prophet > SBEAMS (Systems Biology Experiment Analysis Management System).

Example(s) of projects that use the services of the facility:

• Investigation of drosophila immune response to various pathogenic infections using ICAT™ technique.
• Identification of mammalian proteins that interact with viral epitopes using Tandem-Affinity Purification Tag (TAP-tag) technique.
• Comparison of proteins in mouse bronchoalveolar lavage fluid unique to healthy versus disease state using ICAT™ technique.
• Analysis of protein complement and abundance in halobacterial cells subjected to controlled perturbations in oxygen concentration via iTRAQ™ technique.

Nesvizhskii AI, Vitek O, Aebersold R. Analysis and validation of proteomic data generated by tandem mass spectrometry. Nat Methods. 2007 Oct;4(10):787-97

Ranish JA, Brand M, Aebersold R. Using stable isotope tagging and mass spectrometry to characterize protein complexes and to detect changes in their composition. Methods Mol Biol. 2007;359:17-35.

Qi Y, Ranish JA, Zhu X, Krones A, Zhang J, Aebersold R, Rose DW, Rosenfeld MG, Carrière C. Atbf1 is required for the Pit1 gene early activation. Proceedings of the National Academy of Sciences of the USA. 2008 Feb 13

Mirzaei H, McBee JK, Watts J, Aebersold R. Comparative evaluation of current peptide production platforms used in absolute quantification in proteomics. Molecular & Cellular Proteomics. 2008 Apr;7(4):813-23.

Deutsch EW, Lam H, Aebersold R. PeptideAtlas: a resource for target selection for emerging targeted proteomics workflows. EMBO Reports. 2008 May;9(5):429-34.

Picotti P, Lam H, Campbell D, Deutsch EW, Mirzaei H, Ranish J, Domon B, Aebersold R. A database of mass spectrometric assays for the yeast proteome. Nature Methods. 2008 Nov;5(11):913-4. Links

Brusniak MY, Bodenmiller B, Campbell D, Cooke K, Eddes J, Garbutt A, Lau H, Letarte S, Mueller LN, Sharma V, Vitek O, Zhang N, Aebersold R, Watts JD. Corra: Computational framework and tools for LC-MS discovery and targeted mass spectrometry-based proteomics BMC Bioinformatics. 2008 Dec 16;9(1):542.

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			Contact information: Rob Moritz Phone: 206-732-1244